Medicament for liver regeneration and for treatment of liver failure

ABSTRACT

The present invention relates to the use of a compound which inhibits the activity of MKK4 as a medicament for the treatment of a patient suffering from an impaired liver function, to the use of a compound as a medicament for the treatment of liver failure, including acute/fulminant or chronic liver failure and/or for increasing the regeneration of liver tissue in a patient.

PRIORITY CLAIM

This application is a continuation of U.S. patent application Ser. No.14/875,879 filed Oct. 6, 2015, pending, which is a continuation of U.S.patent application Ser. No. 14/009,738 filed Dec. 23, 2013, now U.S.Pat. No. 9,186,381 issued Nov. 17, 2015, which is the national stageunder 35 USC § 371 of Patent Cooperation Treaty Application NumberPCT/EP2012/056481 filed on Apr. 10, 2012, which claims priority under 35U.S.C § 365 and applicable treaties from U.S. provisional applicationSer. No. 61/473,015 filed Apr. 7, 2011, from EP application no11161588.6 filed Apr. 7, 2011, and from EP application no. 11167373.7filed May 24, 2011, the entire disclosures of these applications areincorporated herein by reference.

BACKGROUND ART

The present invention relates to the use of a compound as a medicamentfor the treatment of a patient suffering from an impaired liverfunction, to the use of a compound as a medicament for the treatment ofliver failure, including acute/fulminant or chronic liver failure and/orfor increasing the regeneration of liver tissue in a patient. Also, theinvention relates to the use of the compound to increase the robustnessand regeneration of cultured hepatocytes in vitro to improve cell basedtherapies, e.g. to a process for cultivating hepatocytes in the presenceof the compound, including the use of the cultivating hepatocytes as atransplant, and for hepatocyte transplantation, respectively, into apatient suffering from liver failure. Further, the invention relates tothe use of the compound for the production of the medicament, and to theuse of hepatocytes cultured in vitro in the presence of the compound forthe production of a hepatocyte transplant.

Further, the invention relates to a bio-artificial liver comprisingcutlivated hepatocytes which contain or are contacted by the compoundwhich can be used as a medicament. Further, the invention relates to aprocess for producing hepatocytes which comprise the compound used as amedicament, and to the use of cultivated hepatocytes being contacted bythe medicament for use as a medicament in the treatment of afunctionally impaired liver, for the treatment of liver failure, and/orfor supporting liver regeneration. Liver failure which can be treatedaccording to the invention includes acute and/or fulminant hepatitis dueto infection with hepatotropic viruses, alcohol abuse, obesity, geneticdiseases like Wilson's disease, hemochromatosis, alpha1-antitrypsindeficiency and related conditions. Liver failure which can be treatedaccording to invention also includes all forms of chronic liver failurewith liver cirrhosis induced by e.g. the causes as indicated above.

STATE OF THE ART

To-date, liver failure is treated by transplantation of a donor liver,however there is a severe shortage of donor organs.

WO98/39352, WO99/14226, and U.S. Pat. No. 7,569,575 B2 describe use andsynthesis of locked nucleic acids (LNA).

OBJECTS OF THE INVENTION

It is an object of the invention to provide a medicament suitable forthe treatment of insufficient liver function, e.g. liver failure, and toprovide cultivated hepatocytes, which can be kept in culture for use ina bio-artificial liver, e.g. for use in the purification of blood or fortransplantation into patients with impaired liver function.

GENERAL DESCRIPTION OF THE INVENTION

The invention achieves the objects by the features of the claims, andespecially by providing a compound inhibiting or inactivating themitogen-activated protein kinase kinase 4 (MAP2K4, also termed MKK4).The nucleotide sequence of the mRNA encoding human MKK4 according toaccession No. NM_003010 is given as SEQ ID NO: 1204. Inhibition orinactivation of the activity of MKK4 can be by reduction of theexpression of MKK4, e.g. by RNA interference induced by siRNA,especially shRNA or microRNA hybridizing to the mRNA encoding MKK4, orby inhibition of MKK4 present in a hepatocyte, e.g. by a kinase-specificinhibitor compound like SP600125, myricitine, Genistein, and PD98059.

The invention is based on the finding that the reduction or deletion ofactive MKK4 in hepatocytes, which can be both cultivated hepatocytes invitro and hepatocytes of a liver of an animal or human patient, resultsin increased regeneration of hepatocytes, e.g. in extended cultivationperiods and in an increase of regeneration of a damaged or impairedliver in vivo, e.g. in experimental animals after induction of anexperimental liver failure representing liver failure in a humanpatient. It has been found that the reduction or deletion of active MKK4in hepatocytes can result in an increased proliferative capacity due toan earlier cell cycle entry and in an increased resistance againstapoptosis. In summary, contacting hepatocytes in vivo by the compoundinhibiting or inactivating MKK4 results in an increased survival of micein experimental models of liver failure. Further, contacting culturedhepatocytes in vitro by the compound inhibiting or inactivating MKK4results in extended cultivation periods and in production of cultivatedhepatocytes, which can be used as a medicament, e.g. as a transplant, orwhich can be used as part of a device for the continuous purification ofblood withdrawn from and returned to a patient.

Inactivation or deletion of MKK4 can be obtained by preventing theexpression of functional MKK4 in liver cells, e.g. by inactivating theendogenous gene encoding MKK4, e.g. by insertional mutagenesis of theendogenous gene encoding MKK4, e.g. by inserting a nucleotide sequencecomprising at least one nucleotide, for disruption of the endogenousgene encoding MKK4, by preventing translation of the mRNA encoding MKK4,or by pharmacological means, e.g. by contacting hepatocytes in vivo orin vitro by a compound which inhibits the kinase function MKK4.

Preferably, inactivation of MKK4 is obtained by reduction or preventionof expression of MKK4 by administration of an inhibitory RNA through RNAinterference (RNAi), which is e.g. an oligonucleotide hybridizing to themRNA encoding MKK4, which inhibitory RNA can e.g. be an siRNA, an siRNAor any form of shRNA contained in a microRNA, e.g. a microRNA basedshRNA, an antisense oligonucleotide, or a mixture of these. Preferably,the oligonucleotide hybridizing to the mRNA encoding MKK4 comprises orconsists 19, 21 or 22 nucleotides which are complementary, especiallyunder physiological and cellular conditions, to the mRNA sequenceencoding MKK4, and a second section, e.g. an antisense strand, which iscomplementary in sequence to the first section. From such adouble-stranded siRNA molecule, in a cellular environment, the firstsection is released from the second section and binds to the mRNAencoding MKK4 to induce the degradation of this mRNA or to induceinhibition of translation. Double stranded RNA molecules (siRNAs) whichlater release one section for mRNA targeting can be directly deliveredinto livers or liver cells but can also be contained in shRNAs or miRNAsfrom which the double stranded RNA is later released by enzymaticprocessing though the cellular RNAi machinery. The sequence of theoligonucleotide hybridizing to the mRNA encoding MKK4 to induce itsdegradation or to prevent its translation can be 100% complementary insequence as usually is the case with siRNAs or shRNAs, but also cancontain mismatches as is often the case with endogenous miRNA, e.g.endogenous miR-15b, miR-24, miR-25, and miR-141, which are also includedas compounds for use in the invention, can target MKK4 mRNA with beingonly partially complementary in sequence. In the description, exemplaryoligonucleotide sequences which are hybridizing to the mRNA encodingMKK4 are give, which oligonucleotides can be contained in an siRNA, e.g.as a first section, preferably forming a double-strand with a reversecomplementary second section contained in the siRNA.

It has been found that inactivation of MKK4 activity, preferably byreduction or inhibition of the expression of MKK4 by presence of anoligonucleotide hybridizing to the mRNA encoding MKK4, can be obtainedby contacting hepatocytes in vivo or in vitro with at least oneoligonucleotide specifically hybridizing to the mRNA encoding MKK4.Contacting in hepatocytes the mRNA which encoded MKK4 can be obtained byadministrating to a human or animal patient the RNA hybridizing to themRNA encoding MKK4 using RNAi through siRNAs by transient in vivotransfection of the siRNA, or alternatively by using, e.g. as amedicament, any means of stable delivery of siRNA, e.g. shRNA,especially microRNA based shRNA or antisense oligonucleotides which arehybridizing to the mRNA encoding MKK4, e.g. use of a viral ortransposon-based nucleic acid construct which contains an expressioncassette encoding the shRNA, for transcription of the shRNA from theexpression cassette. The siRNA, or the nucleic acid construct containingan expression cassette encoding the siRNA, is used as a medicament. Thenucleic acid construct can e.g. be a viral vector or atransposon-containing nucleic acid construct additionally encodingtransposase for integrative stable transduction.

Generally, an oligonucleotide hybridizing to the mRNA encoding MKK4 forreducing or preventing the expression of MKK4 in a liver cell is anoligonucleotide having a sequence hybridizing to the mRNA encoding MKK4,especially hybridizing to SEQ ID NO: 1204, under physiologicalconditions, e.g. in the cellular environment of a liver cell. Thesequence can be fully complementary, i.e. be reverse complementary to asection of the mRNA of SEQ ID NO: 1204, or the sequence can havemismatches as it often occurs in microRNA mediated inhibition oftranslation, e.g. the oligonucleotide sequence has a nucleotide sequenceof at least 80%, preferably of at least 85%, more preferably of at least90% or of 95% identity to a reverse complementary section SEQ ID NO:1204, including as examples the endogenous miRNAs miR-15b, miR-24,miR-25, and miR-141.

Preferred inhibitory oligonucleotides, e.g. shRNA, comprise or consistof one or more of the following oligonucleotides: SEQ ID NO: 1 to SEQ IDNO: 1203. For the design of these sequences the DSIR tool for siRNA andshRNA target design (BMC Bioinformatics, 2006 Nov 30; 7(1):520.) with ascore threshold of 70 was used, and therefore all SEQ ID NO: 1 to 1203have score of at least 70. Inhibitory oligonucleotide sequences, andgroups of inhibitory oligonucleotide sequences having higher scores arepreferred. The sequences are given in an order of descending scorevalue, e.g. SEQ ID NO: 1 has the highest score (107.1), and SEQ ID NO:1200, SEQ ID NO: 1201, SEQ ID NO: 1202, and SEQ ID NO: 1203 have thelowest score (70.0 each). SEQ ID NO: 1 to SEQ ID NO: 11 have a score ofat least 100, SEQ ID NO: 12 to SEQ ID NO: 55 have a score of at least95.1, e.g. of 99.8 to 95.1, SEQ ID NO: 56 to SEQ ID NO: 136 have a scoreof at least 90.0, e.g. of 94.8 to 90.0, SEQ ID NO: 137 to SEQ ID NO: 317have a score of at least 85, e.g. of 89.9 to 85.0, SEQ ID NO: 318 to SEQID NO: 593 have a score of at least 80, e.g. of 84.9 to 80.0, SEQ ID NO:594 to SEQ ID NO: 915 have a score of at least 75.0, e.g. of 79.9 to75.0, and SEQ ID NO: 916 to SEQ ID NO: 1203 have a score between 74.9and 70.0. Further, shRNA or microRNA molecules can comprise one of theseoligonucleotides which are complementary to the mRNA encoding MKK4, e.g.comprising one of these oligonucleotides as a first section and acomplementary second section in the siRNA as hybridizing sections in amicroRNA.

The oligonucleotides hybridizing to the mRNA encoding MKK4 for use as amedicament for the regeneration of liver tissue, or for the treatment ofliver failure, liver insufficiency and/or liver cirrhosis, canpreferably be in the form of RNA, DNA, or hybrids of DNA and RNA,peptide-linkage nucleic acids (PNA), and nucleic acid derivativescontaining a ribose moiety with substituents bridging the 2′-carbon atomand the 4′-carbon atom, e.g. by an oxymethylene group or anaminomethylene group, which derivatives are termed locked nucleic acids(LNA), including further derivatives of the phosphate-sugar backbone,single-stranded, preferably double-stranded, which by intracellularprocessing by the RNAi enzymatic machinery release a single strandedoligonucleotide for hybridization to the mRNA encoding MKK4.

In the alternative to consisting of the use of a nucleic acid sequencehybridizing to the mRNA encoding MKK4, e.g. for direct use as amedicament, the inhibitory RNA can be contained as a coding sequenceunder the control of a promoter in an expression cassette. Depending onthe promoter, which can be a constitutive or an inducible promoter, uponintroduction into the hepatocyte the inhibitory RNA is produced bytranscription.

For introduction of nucleic acid constructs reducing or deleting theexpression of active MKK4, e.g. nucleic acid constructs which interruptthe endogenous gene encoding MKK4 of a hepatocyte or inhibitory RNAhybridizing to the mRNA encoding MKK4, the nucleic acid constructs arepreferably provided in the form of one or more oligonucleotides in apharmaceutically acceptable carrier formulation or in the form of aviral vector packaged in a viral particle or in a virus-like particle. Aviral vector can be a retroviral, a lentiviral vector, anadeno-associated viral vector, or adenoviral vector.

A formulation of the compounds or compositions of the invention forinhibiting or inactivating MKK4 in a pharmaceutically acceptable carriercan e.g. be in a formulation of lipid nanoparticles (LNP) (as e.g.available from Alnylam Pharmaceuticals, USA), a liposome formulation,and/or in a formulation containing a combination with at least onetransfection enhancing agent, e.g. lipofectamine and/or as a Calciumcomplex.

In the alternative or in addition to an oligonucleotide having asequence hybridizing to the mRNA encoding MKK4, e.g. an RNAi hybridizingto the mRNA encoding MKK4, agents inactivating the activity of MKK4,e.g. agents blocking the formation of MKK4 protein, can be used ascompounds for use as a medicament according to the invention. Exemplarycompounds suitable for inactivating MKK4 are e.g. SP600125, myricitine,Genistein, and PD98059, especially for use as medicaments for thetreatment of liver failure, and for the regeneration of liver tissue,respectively.

Histologic analysis of mouse livers with stable knock down of MKK4,experimentally generated by transfection with a nucleic acid constructcontaining an expression cassette for shRNA hybridizing to the mRNAencoding MKK4 showed normal histology. Further, no increase in neoplasmswas detected in the experimental animals, indicating that deletion ofMKK4 does not augment the risk of cancer development.

DETAILED DESCRIPTION OF THE INVENTION

The invention is now described in greater detail by way of examples withreference to the figure, which show in

FIG. 1 schematic representations of nucleic acid constructs forproducing inhibitory RNA,

FIG. 2 a schematic representation of a transposase mediated intrahepatictransfer of an expression cassette encoding an inhibitory RNA (micro RNAbased shRNA) of the invention,

FIG. 3 the time course of the body weight of mice after stabletransposon mediated intrahepatic transfer of an expression cassette forinhibitory RNA and controls, whereas an increase in body weightcorrelates with an increase in liver repopulation with the construct,

FIG. 4 GFP-imaging of explanted mouse livers in the process ofrepopulation by hepatocytes stably expressing shRNA specific forinactivating MKK4,

FIG. 5 a Western blot specific for MKK4 of liver samples of mice stablytransfected with an expression cassette encoding shRNA specific forinactivating MKK4,

FIG. 6 immunofluorescence analysis of the livers of mice stablytransfected with expression cassette encoding shRNA specific forinactivating MKK4,

FIG. 7 a Western blot for cyclin A and E of nuclear liver extracts oftransfected mouse livers in the indicated time course after partialhepatectomy indicating earlier cell cycle entry of hepatocytes stablyexpressing shRNA specific for MKK4,

FIG. 8 a Ki67 staining of mouse livers expressing shRNA specific forMKK4 or control shRNA at the indicated time points after partialhepatectomy,

FIG. 9 a quantifying graph of Ki67 positive hepatocytes depicted in FIG.8,

FIG. 10 TUNEL (upper panel) and H&E staining (lower panel) on liversections after induction of an acute/fulminant liver failure in controlshRNA transfected hepatocytes in comparison to hepatocytes expressing anshRNA specific for RNA of MKK4, which are protected,

FIG. 11 a quantifying graph of apoptotic hepatocytes according to TUNELstaining as depicted in FIG. 10,

FIG. 12 a survival curve of mice expressing the shRNA inactivating MKK4(shMKK4) compared to control mice (shCtr.) after induction of liverfailure,

FIG. 13 a quantifying graph of EdU incorporation into cultured murinehepatocytes with inactivated MKK4 activity (FAHIG-shMKK4) and controlhepatocytes (FAHIG-shCtr) with an inset showing phase contrastmicrographs of these hepatocytes,

FIG. 14 phase contrast micrographs of cultured hepatocytes withinactivated MKK4 (shMKK4) at day 29 (d29) and of control hepatocytes(shCtr) at day 12 (d12), and at day 3 of hepatocytes replated at day 15(replating),

FIG. 15 a survival curve of FAH −/− mice after transplantation ofhepatocytes kept one week in culture expressing shRNA specificallyinactivating MKK4 or a control shRNA,

FIG. 16 photographs, GFP-imaging, α-FAH immunostaining and H&E stainingof liver of mice aged for 1 year following transplantation ofhepatocytes stably expressing shRNA specifically inactivating MKK4, andin

FIG. 17 an overview of the inhibitory effect of preferred smallinhibitory compounds.

Using mice and murine liver tissue and hepatocytes as examples,especially representing human patients and human liver tissue and humanhepatocytes, respectively, it was found that liver regeneration could beincreased by inactivating MKK4 activity both in vivo and in culturedhepatocytes. Mice harbouring livers with reduced MKK4 activity showincreased regenerative capacity under conditions of liver failure, whichalso resulted in an increased survival. Inactivation of MKK4 activitycould efficiently be achieved by inhibitory RNA present in hepatocytes,in vivo and in vitro, which inhibitory RNA could be generated bytranscription from a stably or transiently transfected nucleic acidconstruct containing an expression cassette encoding at least one RNAwhich under physiological conditions hybridizes to the mRNA of MKK4.

Alternatively, the inhibitory RNA could be introduced, e.g. transfectedinto hepatocytes in vivo and in culture, e.g. in the form of an siRNA,shRNA or microRNA, preferably in a suitable formulation, e.g. formulatedas a liposome preparation or a lipid nanoparticle preparation. In thealternative to the use of inhibitory RNA for use as a medicament for thetreatment of liver and hepatocytes, SP600125, myricitine, Genistein,PD98059,3-(Dimethylamino)-N-[3-[(4-hydroxybenzoyl)-amino]-4-methylphenyl]benzamide(ZM 336372),2-hydroxy-1-methyl-4-oxo-N-pyridin-4-ylquinoline-3-carboxamide(BAS00525963), 2-(1H-indazol-5-yliminomethyl)-6-nitrophenolate(BAS00697444),5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-7-oxo-N-phenyl-1H-pyrazolo[1,5-a]pyrimidine-3-carboxamide(SYN22174524),5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-3-(4-fluorophenyl)-1H-pyrazolo[1,5-a]pyrimidin-7-one(SYB22174787),5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-3-(4-methylphenyl)-1H-pyrazolo[1,5-a]pyrimidine-7-one(SYN22175977),3-(4-chlorophenyl)-5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(methoxymethyl)-1H-pyrazolo[1,5-a]pyrimidine-7-one(SYN22176267),-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(methoxymethyl)-3-(4-methylphenyl)-1H-pyrazolo[1,5-a]pyrimidine-7-one(SYN22176367),5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(3-methoxyphenyl)-3-methyl-pyrazolo[5,1-b]pyrimidine-7-ol(SYN22176842),5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-(2-methoxyphenyl)-3-methyl-pyrazolo[5,1-b]pyrimidine-7-ol(SYN22176990),3-(4chlorophenyl)-5-[2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]-2-methyl-1H-pyrazolo[1,5-a]pyrimidine-7-one(SYN22177890),5-amino-3-[(Z)-1-cyano-2-[3-[(4-methoxy-6-piperidin-1-yl-1,3,5-triazin-2-yl)oxy]phenyl]ethenyl]-1-(2-hydroxyethyl)pyrazole-4-carbonitrile(BAS00896568), 2-(1H-indazol-5-yliminomethyl)-6-methoxy-4-nitrophenolate(BAS00697462), 7-oxobenzo[e]perimidine-4-carboxylic acid (BAS00368055),the further compounds contained in Table 1 given herein, andcombinations thereof could be used as medicaments, the presence of whichinactivated MKK4 activity at least partially, also resulting in anincrease of hepatocyte proliferation, protection against inducedapoptosis, and restoration of liver function. These compounds havingspecific inhibitory activity against MKK4 are collectively referred toas small inhibitory compounds. Accordingly, both the inhibitory RNAhaving specificity for the RNA encoding MKK4 and the small inhibitorycompounds having specificity for MKK4 protein each inhibit MKK4 and aretherefore used as medicaments in the treatment of liver failure and/orfor the protection of hepatocytes against apoptosis and/or for theregeneration of hepatocytes. The small inhibitory compounds can beformulated in a pharmaceutically acceptable formulation, comprising e.g.buffer substance and carrier substance as well as formulation additivesas known to the pharmacist, e.g. for i.v., i.m., intra-liveradministration or oral administration.

During functional in vitro testing of hepatocytes containing nucleicacid constructs with stable expression of FAH, GFP and shRNA,hepatocytes were isolated from moose livers and cultivated. It was foundthat only hepatocytes which were transfected with an expression cassetteencoding an inhibitory RNA targeting, i.e. specifically hybridizingwith, the mRNA encoding MKK4 could be cultivated for extended periods,e.g. for over 30 days. In addition, these hepatocytes could betrypsinized and replated according to standard methods.

Transplantation of primary hepatocytes expressing the shRNA against MKK4after 1 week in culture into FAH knock-out mice showed the capacity ofthe hepatocytes in which MKK4 was inactivated to repopulate the liver ofthese mice and allow survival. In contrast, this result could not beobtained by primary hepatocytes expressing the shRNA against MKK4 do notundergo major dedifferentiation during the time of culture.

Example 1 Inactivation of MKK4 by Transcription of Inhibitory RNA Froman Expression Cassette Integrated Into Liver Tissue

The introduction of inhibitory RNA into hepatocytes, i.e. into the liverof a patient, for inactivating MKK4 in vivo by expression of theinhibitory RNA from a nucleic acid construct encoding the inhibitory RNAin an expression cassette is shown on the example of mice (C57BL/6)using an expression cassette encoding the inhibitory RNA for productionof the shRNA hybridizing in the mRNA encoding MKK4. The promotercontrolling transcription of the inhibitory RNA was constitutive.

in short, homozygous FAH-negative mice (FAH −/−) were kept with constantadministration of NTBC in order to block the 4-hydroxyphenylpyruvatedioxygenase which would otherwise lead to the accumulation of toxicmetabolites in the liver. As inhibitory RNA, SEQ ID NO: 1 or,alternatively, SEQ ID NO: 2 was used, both of which hybridize with themRNA encoding MKK4. Each inhibitory RNA was introduced by contacting theliver cells in vivo with nucleic acid constructs withtransposase-specific inverted repeat sections (IR) on both termini,containing an expression cassette for FAH for complementation of the FAH−/− genotype upon expression, by hydrodynamic tail vein injection incombination with a second nucleic acid construct encoding transposasesleeping beauty 13 (SB13) under the control of the PGK promoter.

The nucleic acid constructs are shown in FIG. 1. FIG. 2 schematicallyshows the steps of the genetic manipulation. A first control constructp/T-FAHIG contains the complementing FAH expression cassette and a greenfluorescent protein (GFP) expression cassette comprising the GFPencoding sequence under the control of an IRES element, but encodes noinhibitory RNA. A sequence encoding an inhibitory RNA with no target asa control, which in addition to the GFP expression cassette in 3′ to theGFP encoding sequence encodes a microRNA was contained in the constructp/T-FAHIG-shCtr. A sequence encoding an inhibitory RNA according to theinvention was contained in construct p/T-FAHIG-shMKK4, which in additionto the GFP expression cassette in 3′ to the GFP encoding sequenceencodes a microRNA (depicted as a loop) comprising an shRNA as anexample for an inhibitory RNA. In the example, SEQ ID NO: 1,alternatively SEQ ID NO: 2 was used as a preferred representative ofinhibitory RNA sequences. Following introduction of the nucleic acidconstructs, mice were kept in the absence of NTBC for selecting animalshaving complemented hepatocytes. In cotransfected cells, the transientexpression of SB13 leads the stable integration of the expressioncassette in the genome.

Analyses of mice after introduction of the nucleic acid constructsconfirmed stable transcription of the inhibitory RNA from the nucleicacid construct. In detail, analysis of body weight of mice of FIG. 3shows that the animals having received the control construct p/T-FAHIG(5) as well as the animals having received the control constructp/T-FAHIG-shCtr. (1), which expresses a non-specific RNA could notreconstitute liver function effectively but died.

Animals of those groups having received a nucleic acid constructcontaining an expression cassette for an inhibitory RNA which isspecific for SEQ ID NO: 1, namely p/T-FAHIG-shMKK4.A (2,4) andp/T-FAHIG-shMKK4.B (3) could reconstitute liver function, as shown bythe survival and restoration of body weights.

This result is further supported by FIG. 4 showing livers explanted atday 20 after administration of the nucleic acid construct, where liversare in the process of repopulation by hepatocytes which wereco-transfected in vivo with a nucleic acid construct containing anexpression cassette for FAH and GFP and including an expression cassettefor inhibitory RNA specific for mRNA encoding MKK4 (p/T-FAHIG-shMKK4,both left-hand pictures), or including an expression cassette encoding anon-specific inhibitory RNA (p/T-FAHIG-shCtr, both right-hand pictures).The explanted livers of FIG. 4 show a faster increase of GFPfluorescence over time in vivo from animals co-transfected with thenucleic acid construct which includes the expression cassette encodingan inhibitory RNA specific for the mRNA encoding MKK4 compared toanimals co-transfected with the nucleic acid construct which includesthe expression cassette encoding an inhibitory RNA with no target.

The result from fluorescence is confirmed in this case in fullyrepopulated mouse lives by the immunospecific staining for MKK4 in theWestern blot shown in FIG. 5 and by the immunofluorescence analyses forexpression of MKK4 in the tissue samples of explanted mouse livers whichare shown in FIG. 6.

In FIG. 5, shMKK4-224 denotes protein extracts from mouse liversrepopulated with an expression cassette encoding an inhibitory RNAagainst MKK4 and shMKK4-3553 denotes protein extracts from mouse liversrepopulated with an expression cassette encoding an independentinhibitory RNA against MKK4; tubulin served as a loading control and wasdetected by a specific antibody (α-tub), MKK4 was detected by ananti-MKK4 antibody (α-MKK4). In FIG. 6, FAHIG-shCtr denotes a nucleicacid construct containing the expression cassette for the complementingFAH and for GFP, including a non-specific inhibitory RNA (shCtr).shMKK4-A and shMKK4-B denote nucleic acid constructs containingexpression cassettes for shRNA which specifically hybridize to the mRNAof MKK4.

Both analyses show that only the nucleic acid construct which includesan expression cassette encoding an inhibitory RNA specific for the mRNAencoding MKK4 results in a decrease of MKK4 expression in hepatocytes.

FIG. 7 shows Western blots for cyclin A and E of nuclear extracts fromthe mouse livers contacted with the nucleic acid construct expressingthe shRNA hybridizing to MKK4 mRNA (shMKK4, +) and expressing thenon-specific shRNA (shCtr, +), respectively, at 0, at 24 h, at 38 h, andat 48 h after partial hepatectomy, detected with α-cyclin A antibody(α-Cyc.A) and α-cyclin B antibody (α-CyC.B). This analysis shows thatinactivation of MKK4, which is e.g. obtained by the expression of aninhibitory RNA hybridizing to the mRNA encoding MKK4, leads to anearlier entry of the cell cycle after partial hepatectomy.

FIG. 8 shows a Ki67 stain of livers of the experimental animals havingreceived the nucleic acid construct expressing the inhibitory RNAspecific for MKK4 mRNA (skMKK4) and of animals having received theconstruct expressing the non-specific shRNA (shCtr), respectively, at 0h, 38 h, and 48 h following partial hepatectomy. The analyses show thatthe inactivation of MKK4, which in the example is obtained by presenceof the shRNA which is specific for MKK4 mRNA and is expressed from thenucleic acid construct introduced into the hepatocytes results in anincrease of hepatocyte proliferation in vivo.

FIG. 9 shows a quantification of the Ki67-positive cells from theanalyses of FIG. 8. The increase in hepatocyte proliferation for thehepatocytes containing the shRNA inhibiting expression of MKK4(p/T-FAHIG-shMKK4) is significant in comparison to the non-specificshRNA control (p/T-FAHIG-shCtr).

FIG. 10 shows TUNEL staining for identification of apoptotic cells inliver tissue from mice transfected by an integrating nucleic acidconstruct containing an expression cassette for non-specific shRNA(shCtr.), or an expression cassette for shRNA which specificallyhybridizes to mRNA of MKK4 (shMKK4.224 or shMKK4.355, each expressing amouse-specific siRNA hybridizing to the RNA of MKK4). Apoptosis wasinduced in vivo at 9 h prior to the analysis experimentally by injectionof Jo2 antibody, which interacts with CD95 to induce fulminant liverfailure. TUNEL staining reveals less apoptotic hepatocytes in the livertissue expressing the MKK4-specific shRNA (shMKK4.224, shMKK4.355) thanin controls (shCtr.). The upper row of pictures shows fluorescencemicrographs of TUNEL analyses, the lower row shows bright fieldmicrographs of H&E stained tissue samples.

the quantification of TUNEL analysis following induction of liverfailure is shown in FIG. 11, demonstrating a significantly lower numberof apoptotic hepatocytes in those liver tissues containing the shRNA(shMKK4.224 and shMKK4.3553) that hybridizes to mRNA of MKK4 whencompared to the control with non-specific shRNA (shCtr.).

FIG. 12 shows the survival rate according to Kaplan Meier of micetransfected with the nucleic acid construct expressing the shRNAhybridizing to mRNA of MKK4 (shMKK4.224 and shMKK4.3553) and of controlmice (shCtr.) after the experimental induction of liver failure. Theresult demonstrates that inactivation of MKK4, which inactivation in theexample is obtained by expression of an inhibitory RNA (shRNA) from anexpression of a nucleic acid construct, effectively protects hepatocytesin vivo against apoptosis.

Example 2 Inhibition of MKK4 In Vivo by Transcription of Inhibitory RNAFrom an Expression Cassette Encoding shRNA

For transient transfection of hepatocytes, a nucleic acid constructcontaining or consisting of an expression cassette encoding aninhibitory RNA which specifically hybridizes to the mRNA encoding MKK4,e.g. containing SEQ ID NO: 1 or SEQ ID NO: 2 (which are both specificfor the human and the mouse mRNA of MKK4) was transiently introducedinto hepatocytes. For transient transfection in vivo, the nuclei acidconstruct was formulated in liposomes and administered to theexperimental animals. The liposome formulation contained the lipids3-N-[(qmethoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxy-propylamine)PEG-C-DMA),1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA),1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, in a2:40:10:48 molar per cent ratio.

The effect of increasing proliferation of hepatocytes, protectionagainst induction of apoptosis could be shown as laid out for the stableexpression of the shRNA in Example 1, indicating that the effect waslimited to the period in which the shRNA was present in the hepatocytesusing the analytical methods as described in Example 1. This shows thatMKK4 activity can effectively be inhibited or inactivated by transientexpression of inhibitory RNA, e.g. shRNA or microRNA, from an expressioncassette of a nuclei acid construct which does not integrate into thehepatocyte.

Example 3 Inhibition of MKK4 In Vivo by Inhibitory RNA Transfected IntoHepatocytes

Suitability of inhibitory RNA for use as a medicament could shown bytransfecting the inhibitory RNA which specifically hybridizes to mRNA ofMKK4 into liver tissue in vivo. Inhibitory RNA could be shRNA ormicroRNA, preferably formulated as liposomes or lipid nanoparticles.Generally, reduction or elimination of MKK4 could be obtained in atleast a fraction of the liver tissue contacted by the formulation of theinhibitory RNA using the analytical methods as described in Example 1.This shows that inhibitory RNA specific for mRNA of MKK4 can be used asa medicament, especially for the treatment of impaired liver function.

Example 4 Inhibition of MKK4 In Vivo by SP600125, myricitine, Genistein,or PD98059 in Hepatocytes

In the alternative to inhibition of MKK4 activity in liver tissue byinhibitory RNA, SP600125, myricitine, Genistein, or PD98059 were usedfor inactivating MKK4 in the liver. Generally, SP600125, myricitine,Genistein, or PD98059 were administered to mice as a dosage efficientfor in vivo inactivation of MKK4. Preferably, the dosage was efficientto inactivate at least 80%, more preferably at least 90 or 95% of meanin vivo MKK4 activity.

It could be found that the inactivation of MKK4 in the liver byadministration of SP600125, myricitine, Genistein, or PD98059 as amedicament resulted in a significant increase in liver regeneration, anincrease in proliferation, and in protection against induced apoptosisusing the analytical methods as described in Example 1.

Example 5 Inhibition of MKK4 in Primary Hepatocytes Cultured In Vitro byStable or Transient Transcription of Inhibitory RNA From an ExpressionCassette Encoding shRNA

For in vitro transfection, cultured primary hepatocytes obtained fromexperimental animals were contacted by the nucleic acid construct asdescribed in Example 1 or 2. Generally, the nucleic acid construct couldbe formulated as liposomes according to Example 2.

Generally, stable or transient expression of the inhibitory RNA could beobtained in the cultured hepatocytes, and reduction or elimination ofMKK4 could be detected using the analytical methods as described inExample 1.

For experimental purposes, in the alternative to in vitro transfectionof primary hepatocytes originating from an experimental animal, stablytransfected hepatocytes expressing shRNA specific for MKK4 mRNA wereisolated from the experimental mice generated according to Example 1.Analysis of cultured hepatocytes was by quantification of theincorporation of EdU by primary hepatocytes by flow cytometry. Theresult of cultured transfected hepatocytes after 3 days culture is shownin FIG. 13. The inset phase contrast micrographs and the relation ofhepatocytes containing shRNA specific for mRNA of MKK4, generated byexpression from the transfected expression cassette, show that culturedhepatocytes with inactivated MKK4 (FAHIG-shMKK4) show a drasticallyimproved EdU incorporation as a marker for proliferation over controls(FAHIG-shCtr) without inhibition of MKK4 activity in culture.

Replating of the cultured hepatocytes in fresh culture medium shows theincreased long-term survival of cultured hepatocytes in which MKK4activity is essentially inhibited, e.g. by presence of inhibitory RNA(shMKK4) that specifically hybridizes to mRNA of MKK4, as shown in themicrographs of FIG. 14. Hepatocytes with inactivated MKK4 (shMKK4) canbe cultured effectively at least to day 29 (d29), and can be cultured bytrypsinizing and replating to fresh medium at day 15; right-handmicrographs show day 3 of cells replated after 15 days initial culture.In contrast, transfected cells with a non-specific shRNA (shCtr) show alower long term survival in culture and no growth upon replating after15 days initial culture.

These results show that the inactivation of MKK4 activity drasticallyincreases long term survival and replating efficiency of culturedhepatocytes.

Generally, the generally known Eagles medium was used for hepatocytecultures.

Example 16 Cultured Hepatocytes With Inactivated MKK4 Activity For Useas a Medicament For Liver Regeneration

Hepatocytes from a mouse representing a patient having a compatible oridentical blood group, preferably hepatocytes that were immunologicallycompatible with a later recipient, e.g. a patient, preferably autologoushepatocytes, were cultured. MKK4 activity was inhibited as described inthe above Examples, preferably by transfection of cultured hepatocyteswith a nucleic acid construct containing an expression cassette for aninhibitory RNA hybridizing to the mRNA encoding MKK4, by transfectionwith an inhibitory RNA, preferably repeatedly, or by contacting withSP600125, myricitine, Genistein, or PD98059.

Cultured mouse hepatocytes which stably transfected with a nucleic acidconstruct expressing the complementing FAH and GFP (FAHIG) and aninhibitory RNA specific for the mRNA encoding MKK4 or a non-specificshRNA (Ctrl), respectively, were harvested by trypsinizing. Thesehepatocytes were suspended in a pharmaceutically acceptable carrier andtransplanted into the spleen or liver of FAH −/− mice, whichsubsequently were kept without NTBC. The Kaplan Meier analysis ofsurvival after intraspleenic transplantation of the cultured hepatocytesis shown in FIG. 15. In comparison to mice having received hepatocytescontaining the non-specific shRNA (shRNA.Ctrl) that die at day 37-38(vertical line), mice having received hepatocytes containing shRNA Mkk4specific for the mRNA of MKK4 by expression from the expression cassetteencoding the shRNA have a drastically increased survival.

The experimental FAH −/− mice that had repopulated livers withhepatocytes with an expression cassette for GFP, including the shRNAspecific for the mRNA encoding MKK4 (shRNA.MKK4) were kept for 12 monthsfollowing repopulation. Analyses of explanted livers in bright fieldphotography (Bright), with GFP imaging (GFP) (left-hand pictures of FIG.16) and anti-FAH immunofluorescence and H&E staining of liver section(right-hand pictures of FIG. 16) show no tumor development with stableintrahepatic expression of GFP and of the shRNA specificallyinactivating MKK4. These data emphasize that MKK4 inhibition can be usedto increase regeneration without triggering tumor growth.

Example 7

Cultivated Hepatocytes With Inactivated MKK4 Activity For Use as aDevice For Extracorporal Blood Purification

Cultured hepatocytes obtained as described above, preferably bycultivating primary hepatocytes which were stable transfected with anucleic acid construct expressing an shRNA specific for the mRNAencoding MKK4 were grown on a carrier substrate, e.g. a polymer carrier.The cultured hepatocytes adhering to the carrier substrate were arrangedin a container which was perfused with blood withdrawn from a patient,exemplified by a moose or rat. Blood exiting the container couldimmediately be returned into the patient.

In initial experiments, it could be shown that hepatocytes which aregenetically manipulated to stably express an shRNA inactivating the mRNAencoding MKK4 are stable when grown on a carrier substrate, and thatthese cultures hepatocytes could be used as a blood purification device.

Example 8 Inactivation of MKK4 in In Vitro Analyses

The inhibitory effect of compounds against MKK4 analysed in an in vitroassay using purified MKK4 protein, e.g. obtained from a cell line thatwas genetically manipulated to over-express MKK4 from an expressioncassette containing the nucleotide sequence SEQ ID NO: 1204 as a codingsequence and affinity purification using e.g. an antibody directedagainst MKK4 protein.

In the assay, purified active MKK4 protein was incubated with itssubstrate JNK1a1 and ³²P-labelled gATP (5 μCi, approx. 10 μM), withoutadditional active compound, with the small inhibitory compound, or withGenistein as a positive control. For the assay, kinase assay buffer (20mM HEPES pH 7.5; 10 mM MgCl₂; 1 mg/ml BSA; 1 mM N₃VO₄; 1 mM DDT) wasused. An inhibitory effect of the small inhibitory compound (finalconcentration 50 μM) was detected as a reduction of the phosphorylationactivity of MKK4 protein on its substrate JNK1a1 by measuring the amountof radioactive (³² P) phosphate in JNK1a1. Phosphorylation of JNK1a1 wasmeasured in the presence of 2 ml scintillation cocktail per sample byusing a scintillation counter (Wallac, liquid Scintillation Counter). Inthis assay, Genistein gave an inhibition to approx. 80% activitycompared to the assay without additional active compound.

TABLE 1 small inhibitory compounds assayed for inhibitory activityagainst MKK4: Inhibition, relative activity of MKK4 compared to control(without additional compound) name Structure (%) ZM 336372;3-(Dimethylamino)- N-[3-[(4- hydroxybenzoyl)- amino]-4- methyl-phenyl]benzamide

54.2 BAS00525963; 2-hydroxy-1- methyl-4-oxo-N- pyridin-4- ylquinoline-3-carboxamide

72.6 BAS00697444; 2-(1H-indazol-5- yliminomethyl)-6- nitrophenolate

79.0 SYN22174524; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-7-oxo-N-phenyl-1H- pyrazolo[1,5- a]pyrimidine-3- carboxamide

47.8 SYN22174787; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4-fluorophenyl)-1H- pyrazolo[1,5- a]pyrimidin-7-one

41.5 SYN22175977; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4-methylphenyl)-1H- pyrazolo[1,5- a]pyrimidin-7-one

55.5 SYN22176267; 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-y)ethyl]-2- (methoxymethyl)- 1H-pyrazolo[1,5- a]pyrimidin-7-one

59.1 SYN22176367; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(methoxymethyl)-3- (4-methylphenyl)- 1H-yrazolo[1,5- a]pyrimidin-7-one

49.9 SYN22176842; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(3-methoxyphenyl)-3- methyl- pyrazolo[5,1- b]pyrimidin-7-ol

54.9 SYN22176990; 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(2-methoxyphenyl)-3- methyl- pyrazolo[5,1- b]pyrimidin-7-ol

58.2 SYN22177890; 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)ethyl]-2-methyl- 1H-pyrazolo[1,5- a]pyrimidin-7-one

56.3 BAS00896568; 5-amino-3-[(Z)-1- cyano-2-[3-[(4- methoxy-6-piperidin-1-yl-1,3,5- triazin-2- yl)oxy]phenyl]eth- enyl]-1-(2-hydroxyethyl)py- razole-4-carbonitrile

58.4 BAS00697462; 2-(1H-indazol-5- yliminomethyl)-6- methoxy-4-nitrophenolate

71.1 BAS00368055; 7-oxobenzo[e]- perimidine-4- carboxylic acid

66.2 IUPAC Name: 1- phenyl-2-[[4- phenyl-5-[(5- phenyltetrazol-2-yl)methyl]-1,2,4- triazol-3- yl]sulfanyl]ethanone

IUPAC Name: 2- [[5-[(2,4-dimethyl- anilino)methyl]-4-(furan-2-ylmethyl)- 1,2,4-triazol-3- yl]sulfanylmethyl]-1H-quinazolin-4- one

N-(2-furylmethyl)- N-[1- (isopentylcarbamoyl) ethyl]-5-(morpholinomethyl)- furan-2- carboxamide

IUPAC Name: 4-N- benzyl-1-N-[2-(3,4- dimethoxyphenyl)- ethyl]-4-N-ethylbenzene-1,4- disulfonamide

IPUAC Name: 3-[2- (2,5- dimethoxyphenyl)- 2- oxoethyl]sulfanyl-6-methyl-2H-1,2,4- triazin-5-one

IUPAC Name: 2-[4- (4-methylbenzoyl)- piperidin-1- yl]sulfonylbenzoate

IUPAC Name: 2-[4- [(2,4-dioxo-1,3- thiazolidin-5- ylidene)methyl]-2-methoxyphenoxy]- acetic acid

Popular Name: N- (6-ethoxy-1,3- benzothiazol-2-yl)- 2-[[2-(p-tolyl)-9H-purin-6- yl]sulfanyl]acetamide

2-[4-(4-methoxy- phenyl)-piperazin- 1-yl]-N-(3- morpholino-sulfonyl-phenyl)- acetamide

Popular Name: 5- [[4-[(2,4,6- trioxohexa- hydropyrimidin-5-ylidene)methyl]- phenoxy]methyl]- furan-2-carboxylic

IUPAC Name: 3- (benzimidazol-1- yl)-N-[(2R)-1-[3- (3,4-difluorophenyl)-6- oxopyridazin-1- yl]butan-2- yl]propanamide

IUPAC Name: 2- methyl-3-(pyridin- 3-ylmethyl- amino)benzoate

IUPAC Name: N- [2-[[4-amino-6- (dimethylamino)- 1,3,5-triazin-2-yl]oxy]ethyl]-2-(4- chloro-2- methylphenoxy)acet- amide

IUPAC Name: [3- ethoxy-4-(thiophen- 2- ylmethoxy)phenyl] methyl-(2-morpholin-4-ium-4- ylethyl)azanium

IUPAC Name: 2-[4- (2- hydroxyethyl)pipera- zin-2-yl]-N-(2- pyrrolidin-1-ylsulfonylethyl)pyri- dine-3-carboxamide

IUPAC Name: 1-[[2-(furan-2-yl)- pyrrolo[2,3-b]pyridin-3-yl]methyl-methylamino]-3-(4- methoxyphenoxy)propan-2-ol

IUPAC Name: N- ethyl-3-[2-(4- methoxyphenoxy)eth- oxy]-N- (pyrazolo[1,5-a]pyrimidin-3- ylmethyl)aniline

IUPAC Name: N- ethyl-3-[2-(4- fluorophenoxy)ethoxy]- N-[(2-methylpyrimidin-5- yl)methyl]aniline

IUPAC Name: 2- methoxy-5- morpholin-4- ylbenzoate

IUPAC Name: (1R,2S,3R)-3-(2- aminobenzoyl)-3- methyl-2-N-[(5-methyl-1,2,4- oxadiazol-3- yl)methyl]-1-N- (pyridin-4- ylmethyl)cyclo-propane-1,2- dicarboxamide

IUPAC Name: (2S,3S)-2,3- bis(ethoxycarbonyl) butanedioate

IUPAC Name: diethyl 2-[(1,4- diethoxy-1,4- dioxobut-2-en-2-yl)amino]but-2- enedioate

IUPAC Name: (2S,3S)-2,3- bis(ethoxycarbonyl) butanedioate

IUPAC Name: diethyl 2-[(1,4- diethoxy-1,4- dioxobut-2-en-2-yl)amino]but-2- enedioate

IUPAC Name: 1-ethyl-2-hydroxy-N- (4-hydroxyphenyl)-4- oxoquinoline-3-carboxamide

IUPAC Name: 5- (phenylcarbamoyl- oxy)pentyl N- phenylcarbamate

Popular Name: N- [(5-acetamido-2- methoxy- phenyl)methyl]-2- morpholino-acetamide

IUPAC Name: 4- [4,6-bis(3- carboxypropyl)- 1,3,5-trioxan-2- yl]butanoicacid

IUPAC Name: methyl4- [(4-oxo-2- sulfanylidene-1,3- thiazolidin-5-ylidene)meth- yl]benzoate

IUPAC Name: 2- (3-methylanilino)- N-[(3- nitrophenyl)methyl-ideneamino]acetamide

IUPAC Name: [(2R)-2-[3- [bis[3- (dimethylazaniumyl) propyl]amino]pro-panoyloxy]-3- (dimethylamino)pro- pyl]- dimethylazanium

IUPAC Name: 2-hydroxy-4-oxo-N- pyridin-4-yl-1H-quinoline-3- carboxamide

IUPAC Name: 1-ethyl-2-hydroxy-4-oxo-N-pyridin-4-ylquinoline-3-carboxamide

IUPAC Name: 2,3- bis[2-(2- nitrophenoxy)ethoxy]- 1,4-dioxane

4-hydroxy-2-oxo- N-(4- pyridinylmethyl)- 1,2-dihydro-3- quinolinecarboxamide

IUPAC Name: 7- oxobenzo[e]perimi- dine-4-carboxylic acid

IUPAC Name: 1,3- dioxobenzo[de]iso- quinoline-6- carboxylic acid

IUPAC Name: diethyl(2S)- 2-[[3-[[(2S)-1,5- diethoxy-1,5- dioxopentan-2-yl]amino]-3- oxopropanoyl]amino] pentanedioate

IUPAC Name: diethyl2- [[2-acetamido-3- (4- phenylmethoxyphe-nyl)propanyl]amino] pentanedioate

IUPAC Name: 3- nitor-N[(E)-[3- [(E)-[(3- nitrophenyl)hydrazinyl-idene]methyl]phe- nyl]methylidene amino]aniline

IUPAC Name: (4- methoxyphenyl)meth- ylN-[[4-[2-(3,4-dimethoxyphenyl)eth- ylamino]-4- oxobutan-2- ylidene]amino]carb-

amate IUPAC Name: 3- amino-1,5- dihydropyrimido[5,4- b]indole-2,4- dione

IUPAC Name: 1- [2-(2- fluorophenoxy)eth- yl]-3-[6-[2-(2-fluorophenoxy)eth- ylcarbamoylamino] hexyl]urea

IUPAC Name: methyl4- [[[2-[2-[[4- [hydroxy(methoxy) methyl]phenyl]meth-ylidene]hydrazinyl]- 2- oxoethoxy]acetyl]hydra- zinylidene]meth-yl]benzoate

IUPAC Name: N- [2-(3,4- dimethoxyphenyl)eth- yl]-2-quinolin-8-ylsulfamoylacetamide

IUPAC Name: (2S,3S)- 2,3-bis(4- butoxyphenoxy)- 1,4-dioxane

IUPAC Name: 2-hydroxy- 1-methyl-4-oxo-N-pyridin-4-ylquinoline-3-carboxamide

IUPAC Name: quinoline- 2,4-dicarboxylic acid

IUPAC Name: 2- [(5Z)-5-[(3- hydroxyphenyl)meth- ylidene]-4-oxo-2-sulfanylidene-1,3- thiazolidin-3- yl]propanoic acid

IUPAC Name: diethyl2- acetamido-2-[[5- amino-2-(2-ethoxy- 2-oxoethoxy)phenyl] methyl]propanedioate

IUPAC Name: 4- [2-[2-[2-[(4-amino- 1,2,5-oxadiazol-3-y)oxy]ethoxy]ethoxy] ethoxy]-1,2,5- oxadiazol-3-amine

IUPAC Name: (1R,2S,3S,4S)- 2-(thiophen-2- ylmethylcarbamoyl)bicyclo[2.2.1]hept- 5-ene-3- carboxylate

IUPAC Name: 3- [4-[2-[(4,4- dimethyl-2,6- dioxocyclohexylidene)methylamino]eth- yl]piperazin-1- yl]-1- phenylpyrrolidine- 2,5-dione

IUPAC Name: methyl4- [N-[2-(N-(4- methoxy-4- oxobutanoyl)anilino)ethyl]anilino]-4- oxobutanoate

IUPAC Name: 7- propan-2-ylidene- 2-(pyridin-3- ylmethylcarbamoyl)bicyclo[2.2.1]hept- 5-ene-3- carboxylic acid

1-[4-[2-hydroxy- 3-(2- pyridylmethylamino) propoxy]phenoxy]- 3-(2-pyridylmethylamino) propan-2-ol

IUPAC Name: 3- [5-[(4- hydroxyphenyl)meth- ylidene]-4-oxo-2-sulfanylidene-1,3- thiazolidin-3- yl]propanoic acid

2-(1-benzothiazol- 2 - ylaminoiminoethyl- azo)benzoic

IUPAC Name: 2- [(3-acetylphenyl)- carbamoyl]bicycle- [2.2.1]hept-5-ene-3-carboxylate

IUPAC Name: 2- (1H-indazol-5- yliminomethyl)-6- nitrophenolate

IUPAC Name: 2- bromo-6-[(1H- indazol-5- ylamino)- methylidene]-4-nitrocyclohexa-2,4- dien-1-one

IUPAC Name: 2- (1H-indazol-5- yliminomethyl)-6- methoxy-4-nitrophenolate

N-{2-[2-(1- methyl-4- piperidinylidene)- hydrazino]-2- oxoethyl}-N-(3-nitrophenyl)benzene- sulfonamide (non- preferred name)

IUPAC Name: 4- [5-(naphthalen-1- ylmethylidene)-4- oxo-2-sulfanylidene-1,3- thiazolidin-3- yl]butanoate

IUPAC Name: 5- amino-3-[(Z)-1- cyano-2-[3-[(4- methoxy-6-piperidin-1-yl- 1,3,5-triazin-2- yl)oxy]phenyl]ethe- nyl]-1-(2-hydroxy-ethyl)pyrazole- 4-carbonitrile

IUPAC Name: 8- [2-methoxy-4-[(1- oxo- [1,3]thiazolo[3,2-a]benzimidazol-2- ylidene)meth- yl]phenoxy]-1,3,7- trimethylpurine-2,6-dione

IUPAC Name: 2- [2-[2-(4- methylphenyl)sulfo- nylethoxy]ethoxy]eth- yl4-methylbenzenesulf- onate

IUPAC Name: (2S)-3- acetyl-4-hydroxy- 1-(4- hydroxyphenyl)-2-phenyl-2H-pyrrol- 5-one

IUPAC Name: bis[2-(3,4- dimethylphenyl)-2- oxoethyl] cyclohexane-1,2-dicarboxylate

IUPAC Name: 8- (butoxymethyl)-3- [2-[[5-(2- chlorophenyl)-1H-1,2,4-triazol-3- yl]sulfanyl]acetyl]- 3-methyl-2,7- dioxaspiro[4.4]non-ane-1,6-dione

IUPAC Name: 2- (naphthalen-2- ylsulfonyl- amino)butanoic acid

1-(3,5- dimethoxyphenyl)- N-[(2- nitrophenyl)methyl] methanamine

IUPAC Name: 5- [(2- carboxylatophenyl) sulfamoyl]-2-(3-carboxylatopropyl- amino)benzoate

IUPAC Name: 2- (7H-purin-6- ylazaniumyl)acetate

IUPAC Name: [2- (4-bromophenyl)- 2-oxoethyl]6-(5- methyl-2-oxo-1,3-dihydroimidazol-4- yl)-6-oxohexanoate

3-(2,3-dihydro-1H- indol-1- ylcarbonyl)-1,2,2- trimethylcyclo-pentanecarboxylic acid

IUPAC Name: benzyl-N- [2-[2-[(4-methoxy- 3-nitrophenyl)- methylidene]-hydrazinyl]-2- oxethyl]-N- methylcarbamate

IUPAC Name: methyl4- [[2-[2-[2-[(4- methoxy-4- oxobutyl)amino]-2-oxoethoxy]phenoxy] acetyl]amino]butanoate

IUPAC Name: [2- acetyloxy-4-[2-[5- (ethoxymethyl)-4- imino-2-methylpyrimidin-1- yl]acetyl]phenyl] acetate

IUPAC Name: (4- chloro-2- methylphenyl)meth- ylN-[2-[[4-(dimethylamino)-6- [methoxy(meth- yl)amino]-1,3,5- triazin-2-yl]oxy]ethyl]carba- mate

1-[4-[2-hydroxy-3- (2-nitrophenoxy)- propyl]piperazin-1- yl]-3-(2-nitrophenoxy)- propan-2-ol

IUPAC Name: 2- [(5R)-3-(4- hydroxyphenyl)- 2,4-dioxo-1,3- thiazolidin-5-yl]acetate

IUPAC Name: 2- [2-(2,4- dihydroxyphenyl)- 2- oxoethyl]sulfanyl-4-hydroxy-1H- pyrimidin-6-one

IUPAC Name: 4- hydroxy-2-[2-(1H- indol-3-yl)-2- oxoethyl]sulfanyl-1H-pyrimidin-6- one

IUPAC Name: 3- (3-anilino-2- hydroxypropyl)-1- [[3-(3-anilino-2-hydroxypropyl)- 5,5-dimethyl-2,4- dioxoimidazolidin- 1-yl]methyl]-5,5-dimethylimidazolidine- 2,4-dione

1-(2-furylmethyl)- 4-(3-nitrobenzyl)- piperazine

IUPAC Name: 2- nitro-6-[(5-pyridin- 4-yl-1,3,4- thiadiazol-2-yl)carbamoyl]benzoic acid

IUPAC Name: [2- [[[2-(4- phenylphenoxy)ace- tyl]hydrazinylidene]methyl]phenyl] acetate

2-((4′-HYDROXY- NAPHTHYL)- AZO)BENZOIC ACID

8-hydroxy-5,6- dihydro-4H-11- oxa-6a- azabenzo[de]anthra-cene-7,10-dione

IUPAC Name: 2- (pyridin-3- ylmethylcarbamoyl) bicyclo[2.2.1]hept-5-ene-3- carboxylate

4-Ethyl-5-(4- hydroxyphenyl)- 4H-[1,2,4]triazol- 3-ylsulfanyl]-aceticacid

IUPAC Name: 5- [[4-(benzylamino)- 3- nitrophenyl]sulfonyl-ylamino]benzene- 1,3-dicarboxylate

IUPAC Name: 4- [4-(4-carboxylato- phenoxy)phenyl]- sulfonylphthalate

N-[(2,4- dihydroxyphenyl)- methyleneamino]- 2-[(8-methoxy-2- methyl-4-quinolyl)- sulfanyl]acetamide

IUPAC Name: 1- [2-[2-[2-[2-(2- acetyl- phenoxy)ethoxy]- ethoxy]ethoxy]-phenyl)ethanone

IUPAC Name: [2- [4-(4-chloro-2- nitro- phenoxy)phenyl]- 2-oxoethyl] 2-benzamidoacetate

IUPAC Name: phenacyl 3- [phenacyl- (phenacylamino)- amino]benzoate

IUPAC Name: 1- (4-amino-1,2,5- oxadiazol-3-yl)-5- (1H-benzimidazol- 2-ylsulfanylmethyl)- N-[1-(5-nitrofuran- 2- yl)ethylideneamino]-triazole-4- carboxamide

IUPAC Name: 2- benzamido-N-[1- (furan-2- ylmethylamino)-1- oxopropan-2-yl]benzamide

IUPAC Name: 2- benzamido-N-[1- (3-imidazol-1- ylpropylamino)-1- oxo-3-phenylpropan-2- yl]benzamide

IUPAC Name: ethyl2-[[2- [2-(2,3-dioxoindol- 1- yl)acetyl]oxyacetyl]-amino]-4-methyl- 1,3-thiazole-5- carboxylate

IUPAC Name: ethyl5-[[5- ethoxycarbonyl-3- (2-methoxy-2- oxoethyl)-4-methyl-1H-pyrrol- 2-yl]methyl]-4-(3- methoxy-3- oxopropyl)-3-methyl-1H-pyrrole- 2-carboxylate

IUPAC Name: ethyl2-[[2-(1H- benzimidazol-2- ylsulfanyl)acetyl]-amino]-6-methyl- 5,7-dihydro-4H- thieno[2,3-c]- pyridine-3- carboxylate

keto(3-pyridyl- methylcarbamoyl)- BLAHolate

keto(4- pyridylmethyl- carbamoyl)BLAH- olate

hydroxy-oxo-N-(4- pyridyl)BLAH- carboxamide

IUPAC Name: 3- [[(5R)-2,4-dioxo- 1,3-thiazolidin-5- yl]amino]benzoate

methyl N-acetyl-5- (1,3-dioxo-1,3- dihydro-2H- isoindol-2-yl)-2-(2H-tetrazol-5- yl)norvalinate

IUPAC Name: 3- [(2,3-dioxo-1,4- dihydroquinoxalin- 6-yl)sulfonyl]propan- oate

IUPAC Name: N- [2-(3,4- dimethoxyphenyl)eth- yl]-2-[[5-[(4,6-dimethylpyrimidin- 2-yl)sufanyl- methyl]-1,3,4- oxadiazol-2-yl]-sulfanyl]acetamide

IUPAC Name: (2S)-1- (2,1,3- benzothiadiazol-4- ylsulfonyl)piperidine-2-carboxylic acid

benzyl(veratryl)- BLAH

IUPAC Name: 2- [1-(1,3- benzodioxol-5-yl)- 2,5- dioxopyrrolidin-3-yl]sulfanylpyridine- 3-carboxylate

IUPAC Name: 2- [4-(5-acetyl-1- hydroxy-4- methylimidazol-2-yl)-2-ethoxy- phenoxy]-N-(3- methylphenyl)- acetamide

(3-chlorophenyl)- keto-BLAH- carboxylate

1-naphthyl-oxo- BLAHcarboxylic acid

IUPAC Name: 2- (3,4- dimethoxyphenyl)- 1-[4-(2- fluorophenyl)-piperazin-1- yl]ethanone

IUPAC Name: 4- (4-ethoxyphenyl)- 5-pyridin-4-yl- 1,2,4-triazole-3-thiolate

IUPAC Name: 4- (3-methylphenyl)- 5-pyridin-4-yl- 1,2,4-triazole-3-thiolate

IUPAC Name: ethyl 2-(2-benzyl- sulfonylbenzimid- azol-1-yl)acetate

IUPAC Name: 2- [(3S)-1-(1,3- benzodioxol-5-yl)- 2,5- dioxopyrrolidin-3-yl]sulfanyl- benzoate

Popular Name: 1- [3-(3-methoxy- phenoxy)propyl]-4- [(4-methylphenyl)-sulfonyl]piperazine

IUPAC Name: 3- (pyridin-3- ylmethyl- amino)benzoic acid

2-(4-hydroxy- phenyl)quinoline- 4-carboxylic acid

IUPAC Name: 6- (2-pyridin-4- ylethylcarbamoyl)cyclo- hex-3-ene-1-carboxylate

IUPAC Name: 2- [(3S)-1-(4- hydroxyphenyl)- 2,5- dioxopyrrolidin-3-yl]sulfanylbenzoate

IUPAC Name: 4- (4- methoxyphenyl)-5- pyridin-4-yl-1,2,4-triazole-3-thiolate

IUPAC Name: ethyl5-[(2R)-3- (3,5- dimethylpyrazol-1- yl)-2-hydroxy-propoxy]-1,2- dimethylindole-3- carboxylate

IUPAC Name: 1- naphthalen-2-yl- sulfonylpyrrolidine- 2-carboxylic acid

IUPAC Name: (2S)-2- [(2,3-dioxo-1,4- dihydroquinoxalin- 6-yl)sulfonyl-amino]propanoate

IUPAC Name: (2R)-3- acetyl-4-hydroxy- 1-[2-(1H-indol-3-yl)ethyl]-2-pyridin- 2-yl-2H-pyrrol-5- one

.2.1.0%1,5&]dec- 8-ene-6-carboxylic acid

IUPAC Name: (3S)-3-(1H- indol-3-yl)-3- pyridin-4- ylpropanoic acid

IUPAC Name: 5- methyl-2-pyridin- 4-yl-1H- [1,2,4]triazolo[1,5-a]pyrimidin-7-one

IUPAC Name: (2S)-1-(1- methyl-2- oxobenzo[cd]indol- 6-yl)sulfonylpyrrolidine- 2-carboxylic acid

IUPAC Name: [2- [4-(furan-2- carbonyloxy)phenyl]- 2-oxoethyl]1-(furan-2-ylmethyl)- 5-oxopyrrolidine- 3-carboxylate

IUPAC Name: 4- [2-[5-(4- methoxyphenyl)- 1H-pyrazol-4- yl]ethenyl]-6-(trifluoromethyl)- 1H-pyrimidin-2- one

IUPAC Name: 2-hydroxy-5-[[(E)- (3-methyl-5-oxo-1H- pyrazol-4-ylidene)-methyl]amino]- benzoate

IUPAC Name: 3- oxo-2-(pyridin-4- ylmethyl)-1H- isoindole-4- carboxylicacid

8-hydroxy- [1]benzofuro[3,2- b]quinoline-11- carboxylic acid

IUPAC Name: 5- [[3-methoxy-4- (thiophen-2- ylmethoxy)phenyl]methylamino]-2- morpholin-4- ylbenzoate

oxylic acid

(3R)-4-keto-3- morpholin-4-ium- 4-yl-4-(2-phenoxy ethoxy)butyrate

(3R)-4-keto-3- morpholin-4-ium- 4-yl-4-[[(2S)- tetrahydrofuran-2-yl]methoxy]butyrate

4-oxo-4-[2-[4-(p- tolylsulfonyl)- piperazin-1- yl]ethoxy]butanoic acid

IUPAC Name: 4- (3-chloro-4- fluoropehnyl)-3- pyridin-4-yl-1H-1,2,4-triazole-5- thione

3-[3-(4-pyridyl)-5- thioxo-1H-1,2,4- triazol-4- yl]benzoic

IUPAC Name: 1-(3-phenyl- adamantane-1- carbonyl)pyrrolidine-2-carboxylic acid

IUPAC Name: 4-(2,3-dihydro-1,4- benzodioxin-6-yl)- 3-(3- hydroxyphenyl)-1H-1,2,4-triazole- 5-thione

(dimethylBLAHyl)- methyl

IUPAC Name: N- [1-(3-imidazol-1- ylpropylamino)-3- methyl-1-oxobutan-2-yl]-2- [(4-methoxy- benzoyl)amino]benz- amide

IUPAC Name: 1-ethyl-3-methyl- 2-oxoquinoxaline- 6-carboxylate

IUPAC Name: 3-(4-oxo-2H- pyrazolo[3,4- d]pyrimidin-1- yl)propanoate

3-(benzotriazol-1- yl)-1-[(3R)-3-[4- (3-methylisoxazol-5-yl)-2H-pyrazol- 3-yl]-1-piperidyl]- propan-1-one

IUPAC Name: N-[2-(5-oxo-4- phenyltetrazol-2- yl)ethyl]-2-(4-oxo-3H-phthalazin-1- yl)acetamide

IUPAC Name: ethyl4-[[4- (7-amino-2- methyl- pyrazolo[1,5- a]pyrimidin-5-yl)piperidin-1- yl]methyl]-3,5- dimethyl-1H- pyrrole-2- carboxylate

IUPAC Name: [4- (4-methyl-5- pyrimidin-4-yl-1,3- thiazol-2-yl)piperidin-1-yl]- (1H-1,2,4-triazol- 5-yl)methanone

IUPAC Name: 3- methyl-1-phenyl-5- [(2-pyrrolidin-1- ylsulfonylethyl-amino)methyl]-2H- pyrazolo[3,4- b]pyridin-6-one

1-[2-(4-methoxy- phenyl)pyrimidin- 5-yl]-N-methyl-N-(4,5,6,7-tetrahydro- 1H-indazol-3- ylmethyl)methan- amine

IUPAC Name: N- [[2-(4- methoxy- phenyl)pyrimidin-5- yl]methyl]-N-methyl-1-pyridin- 2-ylpropan-2- amine

IUPAC Name: N- methyl-N-(2- phenoxyethyl)-2- quinazolin-4-yloxyacetamide

IUPAC Name: N- [2-[4-(4- fluorophenyl)-4- hydroxypiperidin-1-yl]ethyl]-2-[(5- methyl-1,3,4- oxadiazol-2- yl)sulfanyl]- acetamide

IUPAC Name: 5- (2-ethoxypyridin- 3-yl)-3-[2-(2- propan-2- ylpyrrolo[2,3-b]pyridin-1- yl)ethyl]-1,2,4- oxadiazole

IUPAC Name: N- [1-(3,4-dihydro- 2H-1,5- benzodioxepin-7- yl)-2-methylpropyl]-6- (2-hydroxyethyl)- pyrazolo[1,5- a]pyrimidine-3-carboxamide

IUPAC Name: N- [2-(1-methyl- benzimidazol-2- yl)ethyl]-2-[2-(3-methyl-1,2,4- oxadiazol-5- yl)phenoxy]- acetamide

2-amino-5-[2-[4- [3-(2,3-dimethyl- phenoxy)propyl]- piperazin-1-yl]-2-oxo-ethyl]-6- methyl-3H- pyrimidin-4-

IUPAC Name: N- [1-(3,5-dimethyl- pyrazol-1- yl)propan-2-yl]-1- (1,5-dimethylpyrazol-4- yl)sulfonylpiperidine- 4-carboxamide

IUPAC Name: 4- [(3,5-dimethyl- pyrazol-1- yl)methyl]-N-[2-(4-fluorophenoxy)- phenyl]-5-methyl- 1,2-oxazole-3- carboxamide

IUPAC Name: 2-(3-methyl-2,6- dioxopurin-7-yl)- N-[1-(7-methyl-1H-indol-3- yl)propan-2- yl]acetamide

IUPAC Name: 7-bicyclo[4.1.0]- heptanyl-[4-[3-(2- methoxyphenoxy)-propyl]piperazin-1- yl]methanone

IUPAC Name: 5- [2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-7-oxo-N-phenyl-1H- pyrazolo[1,5- a]pyrimidine-3- carboxamide

IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-3-(4-fluoro-phenyl)-1H-pyrazolo- [1,5-a]pyrimidin- 7-one

IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-(4-methylphenyl)-1H- pyrazolo[1,5-a]- pyrimidin-7-one

IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-2-(methoxy-methyl)-3-phenyl- 1H-pyrazolo [1,5-a]pyrimidin-7- one

IUPAC Name: 3-(4-chlorophenyl)-5- [2-(3,5-dimethyl-1H-pyrazol-4-yl)ethyl]- 2-(methoxymethyl)- 1H-pyrazolo[1,5-a]-pyrimidin-7-one

IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol- 4-yl)ethyl]-2-(methoxymethyl)- 3-(4-methylphenyl)- 1H-pyrazolo [1,5-a]pyrimidin-7- one

2-(3,4-dimethoxy- phenyl)-5-[2-(3,5- dimethyl-1H- pyrazol-4-yl)ethyl]pyrazolo- [5,1-b]pyrimidin-7- ol

5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(3- methoxyphenyl)-3-methyl- pyrazolo[5,1- b]pyrimidin-7-ol

5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(2- methoxyphenyl)-3-methyl- pyrazolo[5,1- b]pyrimidin-7-ol

5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-3-methyl- 2-(2-thienyl)pyrazolo[5,1- b]pyrimidin-7-ol

IUPAC Name: 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)-ethyl]-2-methyl-1H- pyrazolo[1,5-a]- pyrimidin-7-one

IUPAC Name: 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)- ethyl]-2-methyl-3-phenyl- 1H-pyrazolo[1,5-a]- pyrimidin-7-one

5-[2-(3,5-dimethyl- 1H-pyrazol-4- yl)ethyl]-2-(4- fluorophenyl)-pyrazolo[5,1- b]pyrimidin-7-o

IUPAC Name: 3-(4-chlorophenyl)- 5-[2-(3,5-dimethyl- 1H-pyrazol-4-yl)-ethyl]-1H-pyrazolo- [1,5-a]pyrimidin- 7-one

Compounds of Table 1 can be found on http:\\zinc.docking.org.

FIG. 17 gives an overview of the inhibitory effects of these smallinhibitory compounds on MKK4 protein in relation to the inhibition byGenistein.

In vitro testing according to Example 4 and in vivo testing according toExample 6 could show that these small inhibitory compounds are suitablefor use as a medicament for the treatment of liver failure and/or forthe protection of hepatocytes against apoptosis and/or for theregeneration of hepatocytes.

What is claimed is:
 1. A method of treatment of liver failure and/or forthe treatment of impaired liver function and/or for the protection ofhepatocytes against apoptosis and/or for the regeneration ofhepatocytes, the method comprising a step of administering to a patientin need of treatment a compound, which is an inhibitor of the activityof MKK4, wherein said MKK4 is encoded by mRNA with SEQ ID NO: 1204,wherein the compound is not SP600125, myricitine, PD98059, genistein, orZM
 336372. 2. The method according to claim 1, wherein the compound isat least one of the following:


3. The method according to claim 1, wherein the compound is formulatedas liposomes or lipid nanoparticles.
 4. The method according to claim 1,wherein the patient is a human patient.
 5. A cultivated hepatocyte fortreatment of liver failure and/or for treatment of impaired liverfunction and/or for blood purification, characterized in that thehepatocyte contains a compound which is an inhibitor of the activity ofMKK4, wherein said MKK4 encoded by SEQ ID NO: 1204 and the compound isnot SP600125, myricitine, PD98059, genistein, or ZM
 336372. 6. Amedicament comprising the cultivated hepatocyte of claim
 5. 7. Thecultivated hepatocyte of claim 5, wherein the hepatocyte is arranged ina vessel having an entry port for entry of blood of a patient and anexit port for recirculating the blood to the patient.
 8. The cultivatedhepatocyte of claim 5, wherein the compound is one or more of thecompounds of claim 2.